Theriogenology最新文献

{"title":"Antioxidant effects and compatibility of zinc oxide nanoparticles during in vitro maturation of bovine oocytes and subsequent embryo development","authors":"","doi":"10.1016/j.theriogenology.2024.08.033","DOIUrl":"10.1016/j.theriogenology.2024.08.033","url":null,"abstract":"<div><p>Zinc oxide nanoparticles (ZnO-NPs) have garnered significant attention in biological applications due to their known antioxidant properties. However, their potential impact on assisted reproduction techniques remains largely unexplored, particularly in the context of oocyte quality maintenance within <em>in vitro</em> culture systems, where free radicals can exert detrimental effects. This study investigated the effects of incorporating ZnO-NPs to <em>in vitro</em> maturation (IVM) media on the developmental, cryosurvival, and metabolic profiles of bovine embryos. Three concentrations of ZnO-NPs (0, 1.0, and 1.5 μg/mL) were evaluated. We observed, for the first time, that the inclusion of ZnO-NPs at a concentration of 1.0 μg/mL led to a significant increase in the number of embryonic cells (<em>p</em> &lt; 0.05) accompanied by a reduction in reactive oxygen species production (<em>p</em> &lt; 0.05). Notably, ZnO-NPs did not alter embryonic development, cryosurvival rates, or mitochondrial viability. These findings suggested that ZnO-NPs has antioxidant properties and are compatible with bovine oocytes. Consequently, they may serve as promising supplements to the IVM media, potentially enhancing the efficiency of assisted reproduction techniques.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142126741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cryopreservation of canine ovarian tissue by slow freezing and vitrification: Evaluation of follicular morphology and apoptosis rate","authors":"","doi":"10.1016/j.theriogenology.2024.08.032","DOIUrl":"10.1016/j.theriogenology.2024.08.032","url":null,"abstract":"<div><p>In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P &lt; 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142136753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Collagen and collagenases in mare’s endometrium with endometrosis","authors":"","doi":"10.1016/j.theriogenology.2024.08.031","DOIUrl":"10.1016/j.theriogenology.2024.08.031","url":null,"abstract":"<div><p>Equine endometrosis is a degenerative and predominantly fibrotic condition resulting from progressive and irreversible multifactorial causes that influence the endometrium of mare. Tissue remodeling in the equine endometrium occurs as part of the pathogenesis of endometrosis, a process characterized by a shift in extracellular matrix (ECM) components. The relationship between matrix metalloproteinases and their specific inhibitors is crucial for the remodeling process. Collagen play a significant role in maintaining a healthy uterus and may promote fibrotic processes. The aim of this study was to quantify endometrial collagen deposition using picrosirius 25 red (PSR) staining, and to evaluate gene expression of collagen type 2 (<em>COL-2</em>) and 3 (<em>COL-3</em>), matrix metalloproteinases 1 (<em>MMP-1</em>) and 2 (<em>MMP-2</em>), their tissue inhibitor (<em>TIMP-2</em>), and tumor necrosis factor (<em>TNF-α</em>) in the endometrium of mares with different grades of fibrosis. The samples (n = 34) were classified into three categories based on the frequency and distribution of fibrosis-related changes in the endometrium: Category I (healthy endometrium, n = 12), Category II (moderate fibrosis, n = 12), and Category III (severe fibrosis, n = 10). Collagen quantification demonstrate a substantial proportional increase (P &lt; 0.0001) in collagen deposition across Category I (11.72 ± 1.39 %), Category II (17.76 ± 1.29 %), and Category III (24.15 ± 1.87 %). In transcript evaluations, higher <em>COL-2</em> expression was found in Category II than in mares classified as Category I or III. <em>MMP-1</em> showed increased transcript expression in Category II compared to Category III endometrial samples. Higher expression of MMP-2 was detected in Category III than in Category I and II. <em>TIMP-2</em> showed lower mRNA expression in Category III <em>vs</em> Category I and II. However, <em>TNF-α</em> gene expression was higher in Category II than in Categories I and III. This study demonstrates that endometrial evaluation using PSR can play an important role in routine analyses for the detection and objective quantification of collagen in endometrial tissues. Additionally, this study demonstrated through gene expression analysis that <em>MMP-1</em> may be linked to physiological endometrial remodeling. In contrast, <em>MMP-2</em> could be associated with fibrogenesis in the endometrium, which is regulated by the inhibitor <em>TIMP-2</em>. Furthermore, <em>COL-2</em> and <em>TNF-α</em> could be considered as biological markers involved in the progression endometrosis in mares. As such, the results of this study may contribute to the development of future antifibrotic therapies that aim to delay or even reverse the pathological remodeling of the extracellular matrix in the uterus, in addition to optimizing the diagnosis and prognosis of endometrial fibrosis in mares.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro maturation using porcine follicular fluid-derived exosomes as an alternative to the conventional method","authors":"","doi":"10.1016/j.theriogenology.2024.08.030","DOIUrl":"10.1016/j.theriogenology.2024.08.030","url":null,"abstract":"<div><p>Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte <em>in vitro</em> maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 10⁶ particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 10⁷ particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of <em>Gdf9</em> and <em>Bmp15</em> was observed. The developmental rate was the highest upon treatment with 1.1 × 10⁷ particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; <em>P</em> &lt; 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of <em>Oct4</em>, <em>Sox2</em>, <em>Bcl2</em>, <em>Elf4</em>, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of <em>Bax, Hdac1, Hdac6</em>, and <em>Sirt6</em> decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and <em>Hat1</em> were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the <em>in vitro</em> maturation of porcine oocytes and early embryonic development by regulating gene expression.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0093691X24003583/pdfft?md5=3ec3871d35eae886aef5453074fef629&pid=1-s2.0-S0093691X24003583-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte","authors":"","doi":"10.1016/j.theriogenology.2024.08.029","DOIUrl":"10.1016/j.theriogenology.2024.08.029","url":null,"abstract":"<div><p>Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca²⁺, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca²⁺, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca²⁺ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca²⁺ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142098046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of nonpregnant beef cows based on CL size vs. luteal blood perfusion at 21 days after timed artificial insemination","authors":"","doi":"10.1016/j.theriogenology.2024.08.014","DOIUrl":"10.1016/j.theriogenology.2024.08.014","url":null,"abstract":"<div><p>The aim was to evaluate the efficiency of two different ultrasonographic systems, Doppler mode vs. Two-dimensional mode (B mode), to identify the pregnancy status of beef cows and heifers on day 21 (D21) after Timed Artificial Insemination (TAI). The experiment was performed on a commercial beef farm in central-west region of Brazil using 1895 Nelore heifers and cows. All females had ovulation synchronized for a TAI that was performed on D0. Twenty-one days after the TAI, all animals had their ovaries evaluated by ultrasound for pregnancy diagnosis based on the size of the corpus luteum (CL). Using B mode ultrasonography, females without a CL or with a CL ≤ 10 mm in diameter were considered nonpregnant, whereas females with a CL &gt; 10 mm in diameter were considered potentially pregnant. After the B mode examination, the Doppler mode was turned on, and the CL was evaluated by the subjective percentage of blood perfusion in the total area of the CL. Using Doppler mode, females were considered nonpregnant if they had no CL or the CL had ≤25 % of the total area with detectable blood perfusion, whereas animals with &gt;25 % blood perfusion in the CL were considered potentially pregnant. The results for each method (potentially pregnant or nonpregnant) were later compared with the gold standard technique, which was a pregnancy diagnosis on D33 after TAI using ultrasound with visualization of an embryonic heartbeat. The accuracy was determined using the 2 × 2 contingency table approach. The area under the curve using the receiver operating characteristic curve for Doppler mode and B mode were 0.929 and 0.902 (P &lt; 0.01), respectively. There were almost no false negatives (designated non-pregnant but later pregnant at D33) with either technique (0.2 % vs. 0.3 %; P = 0.65 for Doppler mode vs. B mode, respectively). False positives (designated pregnant but non-pregnant on D33) were greater for B mode compared to Doppler (19.1 % vs. 14.0 %; P &lt; 0.01). This resulted in Doppler mode having similar high values as B mode for Negative Predictive Value (99.9 vs. 99.6 %; P = 0.85) and Sensitivity (99.8 vs. 99.7 %; P = 0.86) but there were differences in Specificity (86 vs. 80.9 %; P &lt; 0.01), Positive Predictive Value (88 vs. 84.3 %; P &lt; 0.01), and Accuracy (93.0 vs. 90.4 %; P &lt; 0.01). In conclusion, evaluation of CL blood perfusion by Doppler produced greater accuracy in the early identification of nonpregnant heifers and cows on D21 after TAI than measurement of CL diameter with B mode ultrasound; although both had over 90 % accuracy in identifying pregnant and nonpregnant females.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142135894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation","authors":"","doi":"10.1016/j.theriogenology.2024.08.027","DOIUrl":"10.1016/j.theriogenology.2024.08.027","url":null,"abstract":"<div><p>Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142044651","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cryopreservation of the whole testes of Asian sea bass (Lates calcarifer) and its effects on apoptosis, germ cell-specific gene expression, germ cell transplantability, and DNA methylation","authors":"","doi":"10.1016/j.theriogenology.2024.08.025","DOIUrl":"10.1016/j.theriogenology.2024.08.025","url":null,"abstract":"<div><p>Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (<em>Lates calcarifer</em>), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of <em>dazl</em> decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of <em>nanos2</em> or <em>nanos3</em> between the fresh and cryopreserved testes. Although qRT-PCR showed lower <em>vasa</em> expression in cryopreserved testicular cells, <em>in situ</em> hybridisation showed expressed <em>vasa</em> in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142083582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism","authors":"","doi":"10.1016/j.theriogenology.2024.08.021","DOIUrl":"10.1016/j.theriogenology.2024.08.021","url":null,"abstract":"<div><h3>Background</h3><p>Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24–48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media.</p></div><div><h3>Material and methods</h3><p>Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca²+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca²⁺ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population.</p></div><div><h3>Results</h3><p>After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; <em>P</em> &lt; 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % <em>(P</em> &lt; 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (<em>P</em> &lt; 0.01). Over time Ca²⁺ increased in all treatment groups compared to time 0h.</p></div><div><h3>Discussion and conclusion</h3><p>Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0093691X2400342X/pdfft?md5=864a3516573d1173e1becf55ace534b9&pid=1-s2.0-S0093691X2400342X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142040490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Incubating frozen-thawed buffalo sperm with olive fruit extracts counteracts thawing-induced oxidative stress and improves semen quality","authors":"","doi":"10.1016/j.theriogenology.2024.08.024","DOIUrl":"10.1016/j.theriogenology.2024.08.024","url":null,"abstract":"<div><p>Freezing-thawing procedures and semen manipulation for <em>in vitro</em> fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress.</p><p>Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 10⁶/mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes.</p><p>The treatment with OFE at all concentrations tested increased (<em>P</em> &lt; 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (<em>P</em> &lt; 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (<em>P</em> &lt; 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (<em>P</em> &lt; 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (<em>P</em> &lt; 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (<em>P</em> &lt; 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and <em>in vitro</em> fertilization ability by enhancing sperm antioxidant capacity.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0093691X24003418/pdfft?md5=ca83bd8a146434a92095db86e4403e4f&pid=1-s2.0-S0093691X24003418-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142040523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}